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Effects of galacto-oligosaccharides on growth and gut function of newborn suckling piglets

Effects of galacto-oligosaccharides on growth and gut function of newborn suckling piglets Background: Most research on galacto-oligosaccharides (GOS) has mainly focused on their prebiotic effects on the hindgut, but their beneficial effects on the small intestine (SI) have received little attention. Since jejunum is the important place to digest and absorb nutrients efficiently, optimal maturation of the jejunum is necessary for maintaining the high growth rate in the neonate. Therefore, this study investigates the effect of the early intervention with GOS on the intestinal development of the jejunum. Methods: A total of 6 litters of neonatal piglets (10 piglets per litter; Duroc × Landrace × Large White) with an average birth weight of 1.55 ± 0.05 kg received 1 of 2 treatments based on their assignment to either the control (CON) group or the GOS (GOS) group in each litter. Piglets in the GOS group were orally administrated 10 mL of a GOS solution (reaching 1 g GOS/kg body weight) per day from the age of 1 to 7 d; the piglets in the CON group were treated with the same dose of physiological saline. All piglets were weaned on d 21. On d 8 and 21 of the experimental trial, 1 pig per group from each of the 6 litters was euthanized. Results: The early intervention with GOS increased the average daily gains in the third week (P < 0.05). Decreased crypt depth was also observed in the jejunum of the piglets on d 21 (P < 0.05). The early intervention with GOS increased the jejunal lactase activity on d 8, maltase activity and sucrase activity on d 21 (P < 0.05). In addition, the early intervention with GOS also facilitated the mRNA expression of Sodium glucose co-transporter 1 (SGLT1)ond8 and the mRNA expression of Glucose transporter type 2 (GLUT2) on d 21 (P < 0.05). It was further determined that GOS up-regulated the mRNA expression of preproglucagon (GCG), insulin-like growth factor 1 (IGF-1), insulin-like growth factor 1 receptor (IGF-1R) and epidermal growth factor (EGF). GOS also up-regulated the protein expression of glucagon-like peptide-2 (GLP-2) and EGF in the jejunum of the piglets. Furthermore, it was also found that GOS enhanced the protein expression of ZO-1 and occludin on d 8 (P < 0.05), as well as increased the mRNA expression of TGF-β and decrease the mRNA expression of IL-12 (P < 0.05). Conclusions: These results indicate that GOS have a positive effect on piglet growth performance in addition to decreasing the crypt depth and enhancing functional development in jejunum of suckling piglets. Keywords: Early intervention, Galacto-oligosaccharides, Growth performance, Intestinal development, Jejunum, Suckling piglets * Correspondence: jwang8@njau.edu.cn National Center for International Research on Animal Gut Nutrition, Jiangsu Key Laboratory of Gastrointestinal Nutrition and Animal Health, Laboratory of Gastrointestinal Microbiology, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Tian et al. Journal of Animal Science and Biotechnology (2018) 9:75 Page 2 of 11 Background maternal differences. GOS-90S was obtained from A nutritional strategy to improve the growth perform- Quantum Hi-Tech Biological Co., Ltd. (China), which con- ance of newborn animals is the basis of improving the tained oligosaccharides with a degree of polymerization overall productivity of animals [1]. The jejunum is an (DP) of 2–8 with approximately 90% (w/w)GOS, 8.5%(w/ important part of the intestine in which the efficient w) lactose, and 1.5% (w/w) glucose on dry matter (DM). digestion and absorption of nutrients take place, and the During the 7 d after birth, all piglets in the GOS group maturation of the jejunum is therefore beneficial for were orally administered 10 mL galacto-oligosaccharides maintaining a high rate of growth among neonates (GOS) solution (1 g GOS/kg body weight [9, 10, 15, 16]) [2, 3]. During the neonatal period, the gastrointes- per day. Half of the dose was given at 9:00 and the other at tinal tract of piglets develops rapidly [4]. Dietary nu- 18:00. Similarly, all the piglets in the CON group were trients are essential for gastrointestinal (GI) growth orally administered the same dose of physiological saline. and functional development,and thenutritional sup- The solution was infused into each piglet’s mouth by an port of GI growth and development is a significant injector without a needle with a soft infusion tube with a part of the nursing process [5]. length of 5 cm. To avoid the potential stress after the In recent years, probiotics and prebiotics have gained swallowing, the piglets were put in the nursing pen imme- considerable attention as growth promoters. Galacto-oli- diately. All piglets were weaned on d 21. The piglets had gosaccharides (GOS), a type of prebiotics, contain 2-8 free access to sow milk and water at all times throughout saccharide units, where one of these units is terminal the experimental period. Average bodyweight throughout glucose and the remaining are galactoses and the treatment process was recorded on d 7, 14 and 21. disaccharides comprised of 2 units of galactoses [6, 7]. Health status was monitored daily until 21 d of age, and all GOS are attractive food additives for infant formula be- piglets remained healthy during the experimental period. cause of their capability of modulating the intestinal microbiota, improving intestinal development, enhan- Sample collection and measurement of villus morphology cing mineral absorption, and protecting the intestinal On d 8 and 21 of the experimental trial, 1 pig per group barrier [8, 9]. Previous studies have shown that GOS from each of the 6 litters was euthanized with an intra- could promote the growth of beneficial bacteria and venous overdose of pentobarbital via a catheterized ear improve host health in vitro and in vivo [10–12]. How- vein as described by Moeser et al. [17]. Blood samples ever, the promotion of intestinal development and the were collected from the anterior vena cava into tubes enhancement of intestinal barrier properties by GOS containing sodium heparin and immediately mixed to have been described mainly in vitro using cell models avoid coagulation. Plasma was obtained after centrifuga- such as the human Caco-2 cell line and rodent models tion at 3,000×g for 15 min at 4 °C and then stored at − [13, 14]; very little has been shown about the effects on 80 °C until analysis. The small intestine (SI) was also re- suckling piglets in vivo. Moreover, most in vivo studies moved. The length of the SI was measured after strip- of GOS have been specifically restricted to the cecum ping off the mesentery by holding the intestine vertically and colon but very few data are currently available for against a ruler. The wet weight of the SI was determined the jejunum. It is essential to explore the effect of early after gently squeezing out the intestinal contents and intervention with GOS on the jejunal development of removing the mesentery and fat. suckling piglets. The mid-jejunal samples (midpoint between the We hypothesized that early intervention with GOS pylorus and the ileocecal valve) were preserved in a for- could increase the growth performance and improve the maldehyde and glutaraldehyde mixing fixative. Then the intestinal development of suckling piglets. The present cross-sections of mid-jejunal samples were prepared study was conducted to explore the effects of early inter- using standard paraffin embedding techniques. To meas- vention with orally administrated GOS on the jejunal ure villus height and crypt depth, the samples were sec- morphology, digestive and absorptive functions, and tioned at 6 μm thickness and stained with hematoxylin barrier functions of suckling piglets. and eosin (HE) [18]. Mucosal samples from the proximal jejunum (at 40 cm from the duodenum-jejunum junc- Methods tion) were collected using a glass slide, rapidly frozen in Animals and experimental treatments liquid nitrogen, and stored at − 80 °C for further ana- A total of 6 litters of neonatal piglets (10 piglets in each; lysis. All samples were collected within 20 min after the Duroc × Landrace × Large White) were used in this piglet had been euthanized. The total protein in the mu- study with an average birth weight of 1.55 ± 0.05 kg. cosal was extracted according to the instructions of a Piglets from each litter were equally assigned to either total protein extraction kit (KGP2100; Keygen Biotech, the control (CON) group or the GOS (GOS) group to Nanjing, China). The mucosal protein concentration of receive 1 of 2 treatments in order to account for any the supernatant fractions was then quantified by a Tian et al. Journal of Animal Science and Biotechnology (2018) 9:75 Page 3 of 11 standard bicinchoninic acid (BCA) protein assay (A045– gene β-actin. The CON group was then established as 3, JianCheng Bioengineering Institute, Nanjing, Jiangsu, the control group. The relative expression of the target China). gene mRNA in each group was calculated as follows: ΔCt = Ct (target gene) - Ct(β-actin), and ΔΔCt = ΔCt RNA extraction, cDNA synthesis, and real-time RT-PCR for (treated group) - ΔCt (CON group on d 8). gene expression analysis The frozen jejunal mucosa was homogenized in 1 mL D-lactic acid and diamine oxidase Trizol Reagent (Invitrogen, Carlsbad, CA, USA), and the The levels of diamine oxidase (DAO; Enzyme Commis- total RNA was isolated according to the manufacture’s sion Number (EC) 1.4.3.6) and D-lactic acid were used recommendations. The absorption ratio (260/280 nm) of as the indices of intestinal mucosal injury in the piglets. all the samples was between 1.8 and 2.0, which indicated The levels of D-lactic acid in the plasma were deter- high purity of the RNA. The total RNA was reverse-tran- mined with a D-lactic acid colorimetric assay kit (BioVi- scribed to cDNA using a PrimeScript RT reagent kit with a sion Inc., Milpitas, CA). D-lactic acid in the plasma was gDNA eraser (Takara Biotechnology (Dalian) Co., Ltd.) ac- expressed in terms of mg/L. Diamine oxidase activities cording to the recommended procedures. in the plasma and jejunal mucosa were measured using The primers for the intestinal nutrient transporter an enzymatic spectrophotometric assay as described by genes: sodium glucose co-transporter 1 (SGLT1), glucose Hu et al. [21]. Diamine oxidase activities in the plasma transporter type 2 (GLUT2); the intestinal growth fac- and jejunal mucosa were expressed in terms of units/mL tors: preproglucagon (GCG), insulin-like growth factor 1 and units/mg mucosa, respectively. (IGF-1), insulin-like growth factor 1 receptor (IGF-1R), epidermal growth factor (EGF); the intestinal barrier Disaccharidase activity related genes: zonula occludens-1 (ZO-1), occludin; the The enzymes studied here were lactase (EC 3.2.1.23), su- intestinal immune factors: interleukin-1β (IL-1β), inter- crase (EC 3.2.1.48) and maltase (EC 3.2.1.20). All assays leukin-10 (IL-10), interleukin-12 (IL-12), toll-like receptor were carried out on the homogenates of mucosal tissue 4(TLR4), transforming growth factor-β (TGF-β), tumor obtained by thawing approximately 0.1 g of tissue and necrosis factor-α (TNF-α) and housekeeping genes homogenizing it in 9 mL phosphate buffer saline (PBS, (β-actin and glyceraldehyde phosphate dehydrogenase pH = 7.2) with an ultrasonic homogenizer. The hom- (GAPDH)) are listed in Additional file 1: Table S1. ogenate was then centrifuged (500×g, 10 min at 4 °C), The target genes and housekeeping genes were mea- and the supernatants were collected. The activity levels sured with an Applied Biosystems 7300 Real-Time PCR of the digestive enzymes lactase (Lactase Activity Testing system using a SYBR Premix Ex Taq™ (Tli RnaseH Plus) Kit, No: A082–1), sucrase (Sucrase Activity Testing Kit, qPCR kit (Takara Biotechnology (Dalian) Co., Ltd.) No: A082–2) and maltase (Maltase Activity Testing Kit, according to the manufacturer’s guidelines. The standard No: A082–3) were determined according to the instruc- dilution and samples were assayed in triplicate in a tions of the manufacturer Nanjing JianCheng Bioengin- 20 μL reaction mixture containing 10 μL of SYBR, eering Institute (Nanjing, Jiangsu, China). 0.4 μL 0.2 μmol/L of forward and reverse primer, 6.8 μL nuclease-free water, and 2 μL of 100 ng/μL DNA tem- Intestinal growth factors plate. The cycling conditions were 95 °C for 30 s, The levels of intestinal growth factors (glucagon-like followed by 40 cycles of 95 °C for 5 s and 60 °C for 34 s. peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), The standard curve was also included in each run to insulin-like growth factor 1 (IGF-1), and epidermal determine PCR efficiency. The specificity and efficiency growth factor (EGF)) in the intestinal mucosa were of the selected primers were confirmed by qRT-PCR determined using the ProcartaPlex™ multiplex immuno- analysis and a dilution series of pooled cDNA at a assay kit (Luminex, Austin, USA) according to the temperature gradient (55–65 °C) for primer-annealing manufacturer’s instructions obtained from Affymetrix and subsequent melting curve analysis. The stability of eBioscience (Santa Clara, USA). The results were nor- the housekeeping genes was evaluated by measuring the malized against the total protein concentration for each fluctuation range of the Ct values (Ct values were sample in an inter-sample comparison. obtained by real-time quantitative PCR). Then, the two candidate genes were analyzed by NormFinder software Tight junction protein expressions [19]. The β-actin was finally identified as the housekeep- Tight junction protein expressions of zonula occludens-1 ing gene because no variation in its expression was (ZO-1) and occludin were measured by Western blotting. observed between treatments. The mRNA expression After the protein concentration of supernatant fractions -ΔΔCt levels were calculated using the 2 method [20]. All was quantified by a standard bicinchoninic acid (BCA) the data were normalized to those of the housekeeping protein assay (Pierce, Rockford, IL, USA), the standardized Tian et al. Journal of Animal Science and Biotechnology (2018) 9:75 Page 4 of 11 protein amounts of boiled samples were isolated with a 15% sodium dodecyl sulfate–polyacrylamide gel elec- trophoresis (SDS–PAGE) and electro-transferred onto polyvinylidene difluoride (PVDF) membranes (Merck Millipore). Membranes were blocked in a skim milk TBS buffer (15 mmol/L Tris-HCl, 150 mmol/L NaCl, 10% skim milk; pH 7.4) and incubated overnight at 4 °C with antibodies for occludin (1:1,000; Abcam) or ZO-1 (1:1,000; Invitrogen). After being washed in a phosphate buffer solution with Tween-20 (PBST), the membranes were incubated with an appropriate horserad- ish peroxidase-conjugated secondary antibody (1: 2,000; Fcmacs-Bio, Beijing, China) for 2 h at room temperature. Finally, the PVDF membranes harboring the target bands were visualized through an electrochemiluminescence system (Tanon, Shanghai, China). Subsequently, the mem- branes were re-probed with a β-actin antibody (1:2,000; Cell Signaling) to assess the equality of loading. Band intensities were quantified using ImageJ version 1.47 software (National Institute of Health, American), and the protein expression was normalized with β-actin and Fig. 1 Effects of galacto-oligosaccharides (GOS) on growth expressed as the mean fold change in relation to the con- performance of suckling piglets. Piglets assigned to CON (n = 6) and trol group. GOS (n = 6) received physiological saline and GOS solution for 7 d after birth, respectively. (a) Body weight and (b) Average daily gain of suckling piglets. Significant differences (P < 0.05) among different Statistical analysis aged piglets within each diet group are indicated by different letters Data were analyzed by SPSS 20.0 (IBM, US) and (upper case for the CON group, lower case for GOS group). expressed as means ± SEM or means ± SD. The model *Indicates a significant difference (P < 0.05) between the diets at included the fixed effects of diet, age, associated interac- each age. Values are expressed as means ± SD, CON: control group; tions, and any random errors with respect to a group or GOS: GOS group an individual piglet. Bodyweight (BW) and average daily gain (ADG) were evaluated using the group in each litter as the experimental unit. The other parameters were showed significantly higher ADG (P < 0.05) than those assessed using each slaughtered piglet as an experimen- fed with no GOS in the third week. In addition, the tal unit. The data were evaluated by two-way ANOVA, results with respect to the digestive organs are presented and differences were considered significant at P < 0.05. in Table 1. SI length was significantly affected by diet (P When a significant dietary effect or an interaction < 0.05), and the same tendency was reported for SI between diet and time was observed, the data were weight (P = 0.078); SI weight / BW tended to be affected further analyzed by using one-way ANOVA with Dun- by the interaction between diet and age (P = 0.096). Fur- can’s post hoc test. And a value of P < 0.05 was used to thermore, piglets fed with diets containing GOS showed indicate statistical significance, whereas a P-value be- significantly increased SI length (P < 0.05) than those fed tween 0.05 and 0.10 was considered to indicate a trend with no GOS on d 8. toward significance. Intestinal morphology and intestinal growth factors Results Diet and age had no significant interactive effects on Growth performance and digestive organ indexes villus height and the villus height / crypt depth ratio of The effects of early intervention with GOS on the the jejunum of piglets (P > 0.05). Crypt depth was signifi- growth performance of suckling piglets are illustrated in cantly affected by the interaction between diet and age Fig. 1. BW was significantly (P < 0.05) affected by the age (P < 0.05). Piglets fed with diets containing GOS showed of piglets, and it also tended to be affected by diet (P = significantly less crypt depth (P < 0.05) than those fed 0.099). Piglets fed with diets containing GOS showed with no GOS on d 21 (Fig. 2). higher BW than those fed with no GOS on d 21, but the The effects of early intervention with GOS on the difference was not significant. For ADG, there was a sig- mRNA expression of intestinal growth factors are pre- nificant interaction (P < 0.05) between diet and the age sented in Fig. 3a-d. The mRNA expression of IGF-1, of the piglets. Piglets fed with diets containing GOS IGF-1R, and EGF was affected by interactions between Tian et al. Journal of Animal Science and Biotechnology (2018) 9:75 Page 5 of 11 Table 1 Effects of galacto-oligosaccharides (GOS) on digestive organ indexes of suckling piglets Items d 8 d 21 SEM P-value CON GOS CON GOS Diet Age Diet × Age SI weight, g 79.72 107.32 193.23 197.74 8.66 0.078 < 0.001 0.196 c b a a SI length, m 4.39 4.93 6.64 6.81 0.17 0.041 < 0.001 0.290 SI weight/SI length, g/m 19.03 21.50 29.06 28.86 0.82 0.183 < 0.001 0.121 SI weight/BW, g/kg 26.85 31.92 31.45 31.20 1.53 0.131 0.218 0.096 SI length/BW, m/kg 1.43 1.49 1.08 1.08 0.04 0.553 < 0.001 0.473 Piglets assigned to CON (n = 6), GOS (n = 6) received physiological saline and GOS solution for 7 d after birth, respectively SI small intestinal diet and age (P < 0.05), and the mRNA expression of observed in the plasma D-lactic acid concentration, GCG was affected by diet. Piglets fed with diets contain- plasma DAO, and jejunal mucosa DAO. On d 8, GOS ing GOS showed significantly higher mRNA expression significantly reduced plasma D-lactic acid concentration of GCG, IGF-1, IGF-1R and EGF than those fed with no and DAO activity (P < 0.05), and increased jejunal mu- GOS on d 8 (P < 0.05), and there was also a significantly cosa DAO (P < 0.05). higher mRNA expression of GCG in GOS-fed piglets Figure 5 shows the gene expression and protein than in those fed with no GOS on d 21 (P < 0.05). expression of ZO-1 and occludin in the jejunal mucosa The effects of early intervention with GOS on the pro- of the piglets. Significant dietary effects were observed tein expression of intestinal growth factors are shown in on the occludin mRNA expression levels (P < 0.05), and Fig. 3e-h. The results show that the protein expression a higher mRNA expression of occludin was observed in of GLP-2 and EGF were affected by diet. Piglets fed with piglets fed with GOS (P < 0.05) in contrast to those fed diets containing GOS showed significantly higher pro- with no GOS on d 8 (Fig. 5a-b). As shown in Fig. 5c-e, tein expression of GLP-2 and EGF than those fed with the protein expression of ZO-1 and occludin was af- no GOS (P < 0.05). fected by interactions between diet and age (P < 0.05). The protein expression of ZO-1 and occludin in the Disaccharidase activity and nutrient transporters jejunal mucosa also increased in the GOS group on d 8 As shown in Fig. 4a-c, lactase and sucrase activity were (P < 0.05). In addition, the protein expression of occludin affected by interactions between diet and age (P < 0.05), in the jejunal mucosa of the GOS piglets was higher and maltase activity was affected by diet. More specific- than that of the CON piglets. ally, piglets fed with diets containing GOS showed significantly higher maltase and sucrase activities (P < Intestinal immune factors 0.05) than those fed with no GOS on d 21, while piglets With respect to intestinal immune factors (Fig. 6), no fed with diets containing GOS showed significantly significant interactions between diet and age were higher lactase activity (P < 0.05) than those fed with no observed on the mRNA expression of IL-1β, IL-10, TLR4 GOS on d 8. and TNF-α (P > 0.05). However, significant interactions To assess gene expression related to the nutrient between diet and age were observed on the TGF-β transporters of jejunum, two kinds of genes were assayed mRNA expression levels (P < 0.05), and significant by q-PCR, as shown in Fig. 4d-e. It was demonstrated dietary effects were observed on the IL-12 mRNA that the mRNA expression of SGLT1 and GLUT2 was expression levels (P < 0.05). It was also determined that affected by interactions between diet and age (P < 0.05). GOS increased the mRNA expression of TGF-β (P < Piglets fed with diets containing GOS showed significantly 0.05) and reduced the mRNA expression of IL-12 on d 8 higher mRNA expression of GLUT2 (P < 0.05) than those (P < 0.05). fed with no GOS on d 8, while piglets fed with diets containing GOS showed significantly higher mRNA ex- Discussion pression of SGLT1 (P < 0.05) than those fed with no GOS In the present study, a neonatal piglet model was used on d 21. to study the effects of early intervention with GOS on growth performance and jejunal development during a D-lactic acid, diamine oxidase, tight junction genes, and week-long intervention period. By determining the protein expressions effects of early feeding strategies on the entire suckling The plasma D-lactic acid concentration and DAO activ- stage, the current experiment made it possible to evalu- ity in the suckling pigs are presented in Table 2. Signifi- ate the effects of GOS (from d 1 to d 7) on piglets’ cant interactions between diet and age (P < 0.05) were growth performance, jejunal morphology, disaccharidase Tian et al. Journal of Animal Science and Biotechnology (2018) 9:75 Page 6 of 11 activity, and barrier function at different ages. The re- sults suggested that GOS had significant effects on ADG, SI length, crypt depth, disaccharidase activity, tight junction expression, and gut permeability in suck- ling piglets. In our study, we referred to the dose of GOS used in rodent model to determine the appropriate dose of GOS for piglets. In previous studies, only the effect of in- creased abundance of Bifidobacterium has been reported in mice fed GOS of 0.26 g/(kg·d) [10]. While increased cecal total weight and wall weight have been reported in mice fed GOS of 1 g/(kg·d) [16]. Anthony et al. [15] found that rats fed GOS of 2.5 g/(kg·d) or 5 g/(kg·d) de- creased food consumption levels. Based on the reported results, the dose of 1 g GOS/kg weight was administered in our study. In addition, it has been reported that the natural oligosaccharide content of sows is approximately in the range of 0.05–0.1 g/dL [22]. According to the Ali- zadeh’s[23] research, we estimated that the total oligo- saccharide intake from the sow milk was about 0.3– 0.6 g/d when the piglets received sow milk of 600 mL/d during the 7 d after birth. In our study, piglets eventually intake GOS of 1.37–2.31 g/d (the initial BW of GOS group: 1.52 kg, the BW on d 7 of GOS group: 2.57 kg) with a consideration of the purity of GOS, about 4 times higher than the oligosaccharide intake solely from sow milk by the piglets. Previous studies have shown that the small intestine has demonstrated a significant increase in tissue mass and surface area of absorption in neonatal piglets [24–28]. For instance, the number of mucosal cells reportedly increased by 50% on the first day after birth and doubled on the third day after birth [28]. These studies indicated that the intestine of piglets developed fastest in the early stages of suckling piglets. Furthermore, it has been reported that GOS could increase cecal total weight and wall weight in mice [16]. Therefore, the pur- pose of this study was to improve intestinal development and increase the growth performance in the early stages of suckling piglets by supplementing with GOS. Consistent with our purpose, the SI length was significantly increased in GOS group on d 8. The increased SI length indicated that a significant increase in the area of the digestive and Fig. 2 The jejunal morphology of suckling piglets. Piglets assigned absorption of nutritious substances, thereby improving to CON (n = 6) and GOS (n = 6) received physiological saline and the growth performance of piglets. In addition, there have GOS solution for 7 d after birth, respectively. (a) Representative histological micrographs of jejunum in suckling piglets. The scale bar been several attempts to demonstrate the use of GOS as of jejunal morphology on d 8 was 500 μm, and the scale bar of potential promoters to enhance animal growth [29, 30]. jejunal morphology on d 21 was 200 μm. (b) Villus height, (c) Crypt Along the same lines as these studies, we have observed depth and (d) Villus height: crypt depth radio of jejunal morphology that early intervention with GOS could improve the BW in suckling piglets. Values are expressed as means ± SD. Bars and ADG of suckling piglets, which also consistent with assigned with different lower-case letters indicate a significant difference. CON: control group; GOS: GOS group our purpose. For suckling piglets, intestinal growth factors play a key role in the development of the intestine. For example, the GLP-1, GLP-2, EGF, and IGF-1 proteins Tian et al. Journal of Animal Science and Biotechnology (2018) 9:75 Page 7 of 11 Fig. 3 The expression of jejunal growth factors in suckling piglets. Piglets assigned to CON (n = 6) and GOS (n = 6) received physiological saline and GOS solution for 7 d after birth, respectively. a-d The relative mRNA expression of jejunal growth factors in suckling piglets. The values were -ΔΔCt calculated relative to the expression of β-actin with formula 2 . e-h The concentrations of jejunal growth factors in suckling piglets. Values are expressed as means ± SD. Bars assigned with different lower-case letters indicate significant differences. CON: control group; GOS: GOS group were able to increase the proliferation, differentiation, in the GOS group was higher than that in CON and apoptosis of intestinal epithelial cells [31, 32]. In this group, and the protein concentration of GLP-2 was study, we observed that the expressions of the intestinal consistent with the mRNA expression of the GCG. growth factors differed between GOS and CON The increased mRNA and protein expression of groups on d 8, but not on d 21. But interestingly, the GLP-2 could increase SI length through the stimula- expressions of intestinal growth factors in GOS pig- tion of epithelial cell antiapoptotic actions by activa- lets on d 8 were close to those in CON piglets and tors of the PI3K-Akt pathway [31]. The activation of GOS piglets on d 21. According to previous results, Akt in the intestinal mucosa has also been implicated the concentrations of growth factors in sow milk at in GLP-2-mediated epithelial glucose uptake [31]. In early lactation stage are higher than those at the late addition, IGF-1 has been identified as a major medi- lactation stage. This may cause the growth and devel- ator through which GLP-2 increases intestinal growth opment of the jejunum to reach the plateau stage at [34]. Also, high mRNA and protein expression of the late period of lactation [33]. And our dynamic GLP-2 may also be modulated by nutrient intake, es- change of daily weight gain confirms this speculation. pecially carbohydrate intake [35, 36]. Therefore, we Furthermore, we also observed that the mRNA ex- believe that GOS could improve the growth perform- pression of IGF-1 and GCG (the precursor of gluca- ance of suckling piglets via promoting jejunal devel- gon and other components is encoded by the GCG) opment and increasing carbohydrate intake. Fig. 4 The jejunal disaccharidase activity and mRNA expression of the glucose transport receptors in suckling piglets. Piglets assigned to CON (n = 6) and GOS (n = 6) received physiological saline and GOS solution for 7 d after birth, respectively. (a-c) The brush border enzyme activity of the jejuna in suckling piglets. (d-e) The relative mRNA expression of jejunal nutrient transporter in suckling piglets. The values were calculated -ΔΔCt relative to the expression of β-actin with formula 2 . Values are expressed as means ± SD. Bars assigned with different lower-case letters indicate significant differences. CON: control group; GOS: GOS group Tian et al. Journal of Animal Science and Biotechnology (2018) 9:75 Page 8 of 11 Table 2 Effects of galacto-oligosaccharides (GOS) on D-lactic acid and diamine oxidase (DAO) in suckling piglets . Items d 8 d 21 SEM P-value CON GOS CON GOS Diet Age Diet × Age a b b ab Plasma D-lactic acid, mg/L 13.13 12.42 12.62 12.81 0.15 0.116 0.661 0.010 a b b b Plasma DAO, units/mL 3.67 3.26 3.24 3.25 0.08 0.019 0.008 0.012 b a b b Jejunal mucosa DAO, units/mg mucosa 2.18 2.32 2.20 2.21 0.03 0.017 0.170 0.025 Piglets assigned to CON (n = 6), GOS (n = 6) received physiological saline and GOS solution for 7 d after birth, respectively Since the jejunum is the main organ for nutrient studies, the lactase activity on d 21 was lower than that absorption, we further analyzed jejunal morphology, on d 8, and the sucrase and maltase activities on d 21 disaccharidase activity, and carbohydrate transporters. In were higher than those on d 8. It is known that maltase the present study, early intervention with GOS signifi- and sucrase activities are important markers to evaluate cantly decreased the crypt depth on d 21, but it did not intestinal development [39, 40]. Hence, the increase of affect the villus height and villus height: crypt depth maltase and sucrase activities implied a certain rapid ratio in the jejunum. The most direct factor affecting maturation of the jejunum. In addition, the present study crypt depth was the change in the proliferation rate of showed that early intervention with GOS up-regulated intestinal stem cells [37]. In addition, decreased crypt lactase activity on d 8, and maltase, sucrase activities on d depth indicated that cell proliferation had decreased in 21. Furthermore, the up-regulated lactase activity on d 8, the GOS group. Villus height has been positively corre- and the maltase, sucrase activities on d 21 would promote lated with the number of cells present [37]. In this study, the polysaccharides in sow milk to be degraded into there was no difference in the villus height between the monosaccharides. This is conducive to the body absorbing two groups, which indicated that decreased cell prolifer- and utilizing the carbohydrates, thereby promoting intes- ation did not affect the growth of the jejunum. This may tinal maturity and host growth. These results therefore have been caused by the increased cell differentiation suggested that the piglets in the GOS group could utilize and the decreased cell apoptosis of the jejunum. Further- carbohydrates more efficiently than those in the CON more, disaccharidase activity is related to intestinal group. After hydrolyzation by disaccharidase, the carbohy- morphology. And the disaccharidase activity determines drates in a diet depend on a carbohydrate transporter to suckling piglets’ capacity for carbohydrate digestion and enter the bloodstream. In this study, the mRNA expres- transport. In previous studies, lactase activity was high sions of SGLT1 and GLUT2 were higher in the piglets with at birth but decreased with the age. However, sucrase GOS intervention than those in the piglets without GOS activity and maltase activity were low at birth, but their intervention, indicating an increased glucose transport activity gradually increased with the age until reaching rate of the intestine. Overall, these results indicate that stability [38]. Consistent with the findings of these early intervention with GOS enhances the degradation Fig. 5 The relative mRNA and protein expression of the jejunal tight junction in suckling piglets. Piglets assigned to CON (n = 6) and GOS (n =6) received physiological saline and GOS solution for 7 d after birth, respectively. (a-b) The relative mRNA expression of the jejunal tight junction in -ΔΔCt suckling piglets. The values were calculated relative to the expression of β-actin with formula 2 .(c) The blots of zonula occludens-1 (ZO-1), occludin, and β-actin of the jejunum mucosa in suckling piglets. (d-e) The relative protein expressions of the jejunal tight junction in suckling piglets. The value of protein expression was the ratio of the densitometry units of tight junction protein to β-actin. Values are expressed as means ± SD. Bars assigned with different lower-case letters indicate significant differences. CON: control group; GOS: GOS group Tian et al. Journal of Animal Science and Biotechnology (2018) 9:75 Page 9 of 11 Fig. 6 The jejunal immune function in suckling piglets. Piglets assigned to CON (n = 6) and GOS (n = 6) received physiological saline and GOS solution for 7 d after birth, respectively. (a-f) The relative mRNA expression of jejunal immune factors in suckling piglets. The values were -ΔΔCt calculated relative to the expression of β-actin with formula 2 . Values are expressed as means ± SD. Bars assigned with different lower-case letters indicate significant differences. CON: control group; GOS: GOS group rate of carbohydrates and the glucose transport rate in GOS intervention promoted the maturation of im- suckling piglets by modulating disaccharidase activity and mune function. In addition, several studies have the expression of glucose transport receptors. shown that the strength of the intestinal barrier was A good mechanical barrier can effectively prevent associated with enhanced piglet performance [43, 50]. bacteria, endotoxins, and other harmful substances In our study, the improvement of intestinal barrier from penetrating the intestinal mucosa, and in terms function was accompanied by increased growth per- of infrastructure, it functions as a tight junction be- formance. Therefore, increased barrier function may tween the intact intestinal epithelial cells and other ensure the absorption of nutrients and prevent bac- epithelial cells [41–43]. ZO-1 and occludin are main teria, endotoxins, and other harmful substances from transmembrane and nonmembrane proteins that entering the body through the intestinal mucosa [51]. form intercellular junctions between the epithelial cells [44, 45]. In addition, DAO and D-lactate serve Conclusion as indicators of intestinal integrity, as they are nor- In conclusion, the results obtained in the present mally presented in very small amounts in blood cir- study indicate that the increased in piglet growth culation. Increased plasma D-lactic acid levels and with GOS supplementation was associated with the serum DAO levels reflect changes in intestinal per- changes in expression of the genes and proteins in- meability, suggesting that the intestinal barrier func- volved in gut endocrine and barrier function, glucose tion has been damaged [46, 47]. Many studies have transporter and immune status. Further study is shown that GOS can reduce gut permeability and in- needed to investigate the exact mechanisms by which crease tight junction expression in vivo and in vitro GOS can promote intestinal development in suckling [48, 49]. Consistent with previous research results, piglets. our study shows that early intervention with GOS could improve the protein expression of ZO-1 and Additional file occludin in the jejunal mucosa on d 8. We also ob- served that plasma D-lactate and DAO decreased in Additional file 1: Table S1. Primer sequences for quantitative real-time theGOS group ond 8. Theseresults indicated that PCR analysis. (DOCX 17 kb) early intervention with GOS could enhance the bar- rier function of the jejunum. Furthermore, the im- Abbreviations provement of intestinal barrier function may imply ADG: Average daily gain; BCA: Bicinchoninic acid; DAO: Diamine oxidase; the improvement of intestinal immune function. DM: Dry matter; DP: Degree of polymerization; EGF: Epidermal growth factor; GAPHH: Glyceraldehyde phosphate dehydrogenase; GCG: Preproglucagon; Therefore, we analyzed the mRNA expression of in- GLP-2: Glucagon-like peptide-2; GLUT2: Glucose transporter type 2; flammatory factors. In this study, GOS increased the GOS: Galacto-oligosaccharides; HE: Hematoxylin and eosin; IGF-1: Insulin-like mRNA expression of TGF-β and reduced the mRNA growth factor 1; IGF-1R: Insulin-like growth factor 1 receptor; IL- 10: Interleukin-10; IL-12: Interleukin-12; IL-1β: Interleukin-1β; PBS: Phosphate expression of IL-12 on d 8. IL-12 is a buffer saline; PBST: Phosphate buffer, saline with Tween-2; pro-inflammatory cytokine, and TGF-β is an anti-in- PVDF: Polyvinylidene difluoride; SDS–PAGE: Sodium dodecyl sulfate– flammatory cytokine. According to the results, early polyacrylamide gel electrophoresis; SGLT1: Sodium glucose co-transporter 1; Tian et al. Journal of Animal Science and Biotechnology (2018) 9:75 Page 10 of 11 SI: Small intestine; TGF-β: Transforming growth factor-β; TLR4: Toll-like 11. Maathuis AJ, Heuvel EG, Schoterman MH, Venema K. Galacto- receptor 4; TNF-α: Tumor necrosis factor-α; ZO-1: Zonula occludens-1 oligosaccharides have prebiotic activity in a dynamic in vitro colon model using a (13) C-labeling technique. J Nutr. 2012;142:1205–12. 12. Sosa N, Gerbino E, Golowczyc MA, Schebor C, Gómez-Zavaglia A, Acknowledgements Tymczyszyn EE. Effect of Galacto-oligosaccharides: Maltodextrin matrices on The authors thank the National Center for International Research on Animal the recovery of lactobacillus plantarum after spray-drying. Front Microbiol. Gut Nutrition for financial support. 2016;7:584. 13. Varasteh S, Braber S, Garssen J, Fink-Gremmels J. Galacto-oligosaccharides Funding exert a protective effect against heat stress in a Caco-2 cell model. 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Effects of galacto-oligosaccharides on growth and gut function of newborn suckling piglets

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Life Sciences; Agriculture; Biotechnology; Food Science; Animal Genetics and Genomics; Animal Physiology
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Abstract

Background: Most research on galacto-oligosaccharides (GOS) has mainly focused on their prebiotic effects on the hindgut, but their beneficial effects on the small intestine (SI) have received little attention. Since jejunum is the important place to digest and absorb nutrients efficiently, optimal maturation of the jejunum is necessary for maintaining the high growth rate in the neonate. Therefore, this study investigates the effect of the early intervention with GOS on the intestinal development of the jejunum. Methods: A total of 6 litters of neonatal piglets (10 piglets per litter; Duroc × Landrace × Large White) with an average birth weight of 1.55 ± 0.05 kg received 1 of 2 treatments based on their assignment to either the control (CON) group or the GOS (GOS) group in each litter. Piglets in the GOS group were orally administrated 10 mL of a GOS solution (reaching 1 g GOS/kg body weight) per day from the age of 1 to 7 d; the piglets in the CON group were treated with the same dose of physiological saline. All piglets were weaned on d 21. On d 8 and 21 of the experimental trial, 1 pig per group from each of the 6 litters was euthanized. Results: The early intervention with GOS increased the average daily gains in the third week (P < 0.05). Decreased crypt depth was also observed in the jejunum of the piglets on d 21 (P < 0.05). The early intervention with GOS increased the jejunal lactase activity on d 8, maltase activity and sucrase activity on d 21 (P < 0.05). In addition, the early intervention with GOS also facilitated the mRNA expression of Sodium glucose co-transporter 1 (SGLT1)ond8 and the mRNA expression of Glucose transporter type 2 (GLUT2) on d 21 (P < 0.05). It was further determined that GOS up-regulated the mRNA expression of preproglucagon (GCG), insulin-like growth factor 1 (IGF-1), insulin-like growth factor 1 receptor (IGF-1R) and epidermal growth factor (EGF). GOS also up-regulated the protein expression of glucagon-like peptide-2 (GLP-2) and EGF in the jejunum of the piglets. Furthermore, it was also found that GOS enhanced the protein expression of ZO-1 and occludin on d 8 (P < 0.05), as well as increased the mRNA expression of TGF-β and decrease the mRNA expression of IL-12 (P < 0.05). Conclusions: These results indicate that GOS have a positive effect on piglet growth performance in addition to decreasing the crypt depth and enhancing functional development in jejunum of suckling piglets. Keywords: Early intervention, Galacto-oligosaccharides, Growth performance, Intestinal development, Jejunum, Suckling piglets * Correspondence: jwang8@njau.edu.cn National Center for International Research on Animal Gut Nutrition, Jiangsu Key Laboratory of Gastrointestinal Nutrition and Animal Health, Laboratory of Gastrointestinal Microbiology, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Tian et al. Journal of Animal Science and Biotechnology (2018) 9:75 Page 2 of 11 Background maternal differences. GOS-90S was obtained from A nutritional strategy to improve the growth perform- Quantum Hi-Tech Biological Co., Ltd. (China), which con- ance of newborn animals is the basis of improving the tained oligosaccharides with a degree of polymerization overall productivity of animals [1]. The jejunum is an (DP) of 2–8 with approximately 90% (w/w)GOS, 8.5%(w/ important part of the intestine in which the efficient w) lactose, and 1.5% (w/w) glucose on dry matter (DM). digestion and absorption of nutrients take place, and the During the 7 d after birth, all piglets in the GOS group maturation of the jejunum is therefore beneficial for were orally administered 10 mL galacto-oligosaccharides maintaining a high rate of growth among neonates (GOS) solution (1 g GOS/kg body weight [9, 10, 15, 16]) [2, 3]. During the neonatal period, the gastrointes- per day. Half of the dose was given at 9:00 and the other at tinal tract of piglets develops rapidly [4]. Dietary nu- 18:00. Similarly, all the piglets in the CON group were trients are essential for gastrointestinal (GI) growth orally administered the same dose of physiological saline. and functional development,and thenutritional sup- The solution was infused into each piglet’s mouth by an port of GI growth and development is a significant injector without a needle with a soft infusion tube with a part of the nursing process [5]. length of 5 cm. To avoid the potential stress after the In recent years, probiotics and prebiotics have gained swallowing, the piglets were put in the nursing pen imme- considerable attention as growth promoters. Galacto-oli- diately. All piglets were weaned on d 21. The piglets had gosaccharides (GOS), a type of prebiotics, contain 2-8 free access to sow milk and water at all times throughout saccharide units, where one of these units is terminal the experimental period. Average bodyweight throughout glucose and the remaining are galactoses and the treatment process was recorded on d 7, 14 and 21. disaccharides comprised of 2 units of galactoses [6, 7]. Health status was monitored daily until 21 d of age, and all GOS are attractive food additives for infant formula be- piglets remained healthy during the experimental period. cause of their capability of modulating the intestinal microbiota, improving intestinal development, enhan- Sample collection and measurement of villus morphology cing mineral absorption, and protecting the intestinal On d 8 and 21 of the experimental trial, 1 pig per group barrier [8, 9]. Previous studies have shown that GOS from each of the 6 litters was euthanized with an intra- could promote the growth of beneficial bacteria and venous overdose of pentobarbital via a catheterized ear improve host health in vitro and in vivo [10–12]. How- vein as described by Moeser et al. [17]. Blood samples ever, the promotion of intestinal development and the were collected from the anterior vena cava into tubes enhancement of intestinal barrier properties by GOS containing sodium heparin and immediately mixed to have been described mainly in vitro using cell models avoid coagulation. Plasma was obtained after centrifuga- such as the human Caco-2 cell line and rodent models tion at 3,000×g for 15 min at 4 °C and then stored at − [13, 14]; very little has been shown about the effects on 80 °C until analysis. The small intestine (SI) was also re- suckling piglets in vivo. Moreover, most in vivo studies moved. The length of the SI was measured after strip- of GOS have been specifically restricted to the cecum ping off the mesentery by holding the intestine vertically and colon but very few data are currently available for against a ruler. The wet weight of the SI was determined the jejunum. It is essential to explore the effect of early after gently squeezing out the intestinal contents and intervention with GOS on the jejunal development of removing the mesentery and fat. suckling piglets. The mid-jejunal samples (midpoint between the We hypothesized that early intervention with GOS pylorus and the ileocecal valve) were preserved in a for- could increase the growth performance and improve the maldehyde and glutaraldehyde mixing fixative. Then the intestinal development of suckling piglets. The present cross-sections of mid-jejunal samples were prepared study was conducted to explore the effects of early inter- using standard paraffin embedding techniques. To meas- vention with orally administrated GOS on the jejunal ure villus height and crypt depth, the samples were sec- morphology, digestive and absorptive functions, and tioned at 6 μm thickness and stained with hematoxylin barrier functions of suckling piglets. and eosin (HE) [18]. Mucosal samples from the proximal jejunum (at 40 cm from the duodenum-jejunum junc- Methods tion) were collected using a glass slide, rapidly frozen in Animals and experimental treatments liquid nitrogen, and stored at − 80 °C for further ana- A total of 6 litters of neonatal piglets (10 piglets in each; lysis. All samples were collected within 20 min after the Duroc × Landrace × Large White) were used in this piglet had been euthanized. The total protein in the mu- study with an average birth weight of 1.55 ± 0.05 kg. cosal was extracted according to the instructions of a Piglets from each litter were equally assigned to either total protein extraction kit (KGP2100; Keygen Biotech, the control (CON) group or the GOS (GOS) group to Nanjing, China). The mucosal protein concentration of receive 1 of 2 treatments in order to account for any the supernatant fractions was then quantified by a Tian et al. Journal of Animal Science and Biotechnology (2018) 9:75 Page 3 of 11 standard bicinchoninic acid (BCA) protein assay (A045– gene β-actin. The CON group was then established as 3, JianCheng Bioengineering Institute, Nanjing, Jiangsu, the control group. The relative expression of the target China). gene mRNA in each group was calculated as follows: ΔCt = Ct (target gene) - Ct(β-actin), and ΔΔCt = ΔCt RNA extraction, cDNA synthesis, and real-time RT-PCR for (treated group) - ΔCt (CON group on d 8). gene expression analysis The frozen jejunal mucosa was homogenized in 1 mL D-lactic acid and diamine oxidase Trizol Reagent (Invitrogen, Carlsbad, CA, USA), and the The levels of diamine oxidase (DAO; Enzyme Commis- total RNA was isolated according to the manufacture’s sion Number (EC) 1.4.3.6) and D-lactic acid were used recommendations. The absorption ratio (260/280 nm) of as the indices of intestinal mucosal injury in the piglets. all the samples was between 1.8 and 2.0, which indicated The levels of D-lactic acid in the plasma were deter- high purity of the RNA. The total RNA was reverse-tran- mined with a D-lactic acid colorimetric assay kit (BioVi- scribed to cDNA using a PrimeScript RT reagent kit with a sion Inc., Milpitas, CA). D-lactic acid in the plasma was gDNA eraser (Takara Biotechnology (Dalian) Co., Ltd.) ac- expressed in terms of mg/L. Diamine oxidase activities cording to the recommended procedures. in the plasma and jejunal mucosa were measured using The primers for the intestinal nutrient transporter an enzymatic spectrophotometric assay as described by genes: sodium glucose co-transporter 1 (SGLT1), glucose Hu et al. [21]. Diamine oxidase activities in the plasma transporter type 2 (GLUT2); the intestinal growth fac- and jejunal mucosa were expressed in terms of units/mL tors: preproglucagon (GCG), insulin-like growth factor 1 and units/mg mucosa, respectively. (IGF-1), insulin-like growth factor 1 receptor (IGF-1R), epidermal growth factor (EGF); the intestinal barrier Disaccharidase activity related genes: zonula occludens-1 (ZO-1), occludin; the The enzymes studied here were lactase (EC 3.2.1.23), su- intestinal immune factors: interleukin-1β (IL-1β), inter- crase (EC 3.2.1.48) and maltase (EC 3.2.1.20). All assays leukin-10 (IL-10), interleukin-12 (IL-12), toll-like receptor were carried out on the homogenates of mucosal tissue 4(TLR4), transforming growth factor-β (TGF-β), tumor obtained by thawing approximately 0.1 g of tissue and necrosis factor-α (TNF-α) and housekeeping genes homogenizing it in 9 mL phosphate buffer saline (PBS, (β-actin and glyceraldehyde phosphate dehydrogenase pH = 7.2) with an ultrasonic homogenizer. The hom- (GAPDH)) are listed in Additional file 1: Table S1. ogenate was then centrifuged (500×g, 10 min at 4 °C), The target genes and housekeeping genes were mea- and the supernatants were collected. The activity levels sured with an Applied Biosystems 7300 Real-Time PCR of the digestive enzymes lactase (Lactase Activity Testing system using a SYBR Premix Ex Taq™ (Tli RnaseH Plus) Kit, No: A082–1), sucrase (Sucrase Activity Testing Kit, qPCR kit (Takara Biotechnology (Dalian) Co., Ltd.) No: A082–2) and maltase (Maltase Activity Testing Kit, according to the manufacturer’s guidelines. The standard No: A082–3) were determined according to the instruc- dilution and samples were assayed in triplicate in a tions of the manufacturer Nanjing JianCheng Bioengin- 20 μL reaction mixture containing 10 μL of SYBR, eering Institute (Nanjing, Jiangsu, China). 0.4 μL 0.2 μmol/L of forward and reverse primer, 6.8 μL nuclease-free water, and 2 μL of 100 ng/μL DNA tem- Intestinal growth factors plate. The cycling conditions were 95 °C for 30 s, The levels of intestinal growth factors (glucagon-like followed by 40 cycles of 95 °C for 5 s and 60 °C for 34 s. peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), The standard curve was also included in each run to insulin-like growth factor 1 (IGF-1), and epidermal determine PCR efficiency. The specificity and efficiency growth factor (EGF)) in the intestinal mucosa were of the selected primers were confirmed by qRT-PCR determined using the ProcartaPlex™ multiplex immuno- analysis and a dilution series of pooled cDNA at a assay kit (Luminex, Austin, USA) according to the temperature gradient (55–65 °C) for primer-annealing manufacturer’s instructions obtained from Affymetrix and subsequent melting curve analysis. The stability of eBioscience (Santa Clara, USA). The results were nor- the housekeeping genes was evaluated by measuring the malized against the total protein concentration for each fluctuation range of the Ct values (Ct values were sample in an inter-sample comparison. obtained by real-time quantitative PCR). Then, the two candidate genes were analyzed by NormFinder software Tight junction protein expressions [19]. The β-actin was finally identified as the housekeep- Tight junction protein expressions of zonula occludens-1 ing gene because no variation in its expression was (ZO-1) and occludin were measured by Western blotting. observed between treatments. The mRNA expression After the protein concentration of supernatant fractions -ΔΔCt levels were calculated using the 2 method [20]. All was quantified by a standard bicinchoninic acid (BCA) the data were normalized to those of the housekeeping protein assay (Pierce, Rockford, IL, USA), the standardized Tian et al. Journal of Animal Science and Biotechnology (2018) 9:75 Page 4 of 11 protein amounts of boiled samples were isolated with a 15% sodium dodecyl sulfate–polyacrylamide gel elec- trophoresis (SDS–PAGE) and electro-transferred onto polyvinylidene difluoride (PVDF) membranes (Merck Millipore). Membranes were blocked in a skim milk TBS buffer (15 mmol/L Tris-HCl, 150 mmol/L NaCl, 10% skim milk; pH 7.4) and incubated overnight at 4 °C with antibodies for occludin (1:1,000; Abcam) or ZO-1 (1:1,000; Invitrogen). After being washed in a phosphate buffer solution with Tween-20 (PBST), the membranes were incubated with an appropriate horserad- ish peroxidase-conjugated secondary antibody (1: 2,000; Fcmacs-Bio, Beijing, China) for 2 h at room temperature. Finally, the PVDF membranes harboring the target bands were visualized through an electrochemiluminescence system (Tanon, Shanghai, China). Subsequently, the mem- branes were re-probed with a β-actin antibody (1:2,000; Cell Signaling) to assess the equality of loading. Band intensities were quantified using ImageJ version 1.47 software (National Institute of Health, American), and the protein expression was normalized with β-actin and Fig. 1 Effects of galacto-oligosaccharides (GOS) on growth expressed as the mean fold change in relation to the con- performance of suckling piglets. Piglets assigned to CON (n = 6) and trol group. GOS (n = 6) received physiological saline and GOS solution for 7 d after birth, respectively. (a) Body weight and (b) Average daily gain of suckling piglets. Significant differences (P < 0.05) among different Statistical analysis aged piglets within each diet group are indicated by different letters Data were analyzed by SPSS 20.0 (IBM, US) and (upper case for the CON group, lower case for GOS group). expressed as means ± SEM or means ± SD. The model *Indicates a significant difference (P < 0.05) between the diets at included the fixed effects of diet, age, associated interac- each age. Values are expressed as means ± SD, CON: control group; tions, and any random errors with respect to a group or GOS: GOS group an individual piglet. Bodyweight (BW) and average daily gain (ADG) were evaluated using the group in each litter as the experimental unit. The other parameters were showed significantly higher ADG (P < 0.05) than those assessed using each slaughtered piglet as an experimen- fed with no GOS in the third week. In addition, the tal unit. The data were evaluated by two-way ANOVA, results with respect to the digestive organs are presented and differences were considered significant at P < 0.05. in Table 1. SI length was significantly affected by diet (P When a significant dietary effect or an interaction < 0.05), and the same tendency was reported for SI between diet and time was observed, the data were weight (P = 0.078); SI weight / BW tended to be affected further analyzed by using one-way ANOVA with Dun- by the interaction between diet and age (P = 0.096). Fur- can’s post hoc test. And a value of P < 0.05 was used to thermore, piglets fed with diets containing GOS showed indicate statistical significance, whereas a P-value be- significantly increased SI length (P < 0.05) than those fed tween 0.05 and 0.10 was considered to indicate a trend with no GOS on d 8. toward significance. Intestinal morphology and intestinal growth factors Results Diet and age had no significant interactive effects on Growth performance and digestive organ indexes villus height and the villus height / crypt depth ratio of The effects of early intervention with GOS on the the jejunum of piglets (P > 0.05). Crypt depth was signifi- growth performance of suckling piglets are illustrated in cantly affected by the interaction between diet and age Fig. 1. BW was significantly (P < 0.05) affected by the age (P < 0.05). Piglets fed with diets containing GOS showed of piglets, and it also tended to be affected by diet (P = significantly less crypt depth (P < 0.05) than those fed 0.099). Piglets fed with diets containing GOS showed with no GOS on d 21 (Fig. 2). higher BW than those fed with no GOS on d 21, but the The effects of early intervention with GOS on the difference was not significant. For ADG, there was a sig- mRNA expression of intestinal growth factors are pre- nificant interaction (P < 0.05) between diet and the age sented in Fig. 3a-d. The mRNA expression of IGF-1, of the piglets. Piglets fed with diets containing GOS IGF-1R, and EGF was affected by interactions between Tian et al. Journal of Animal Science and Biotechnology (2018) 9:75 Page 5 of 11 Table 1 Effects of galacto-oligosaccharides (GOS) on digestive organ indexes of suckling piglets Items d 8 d 21 SEM P-value CON GOS CON GOS Diet Age Diet × Age SI weight, g 79.72 107.32 193.23 197.74 8.66 0.078 < 0.001 0.196 c b a a SI length, m 4.39 4.93 6.64 6.81 0.17 0.041 < 0.001 0.290 SI weight/SI length, g/m 19.03 21.50 29.06 28.86 0.82 0.183 < 0.001 0.121 SI weight/BW, g/kg 26.85 31.92 31.45 31.20 1.53 0.131 0.218 0.096 SI length/BW, m/kg 1.43 1.49 1.08 1.08 0.04 0.553 < 0.001 0.473 Piglets assigned to CON (n = 6), GOS (n = 6) received physiological saline and GOS solution for 7 d after birth, respectively SI small intestinal diet and age (P < 0.05), and the mRNA expression of observed in the plasma D-lactic acid concentration, GCG was affected by diet. Piglets fed with diets contain- plasma DAO, and jejunal mucosa DAO. On d 8, GOS ing GOS showed significantly higher mRNA expression significantly reduced plasma D-lactic acid concentration of GCG, IGF-1, IGF-1R and EGF than those fed with no and DAO activity (P < 0.05), and increased jejunal mu- GOS on d 8 (P < 0.05), and there was also a significantly cosa DAO (P < 0.05). higher mRNA expression of GCG in GOS-fed piglets Figure 5 shows the gene expression and protein than in those fed with no GOS on d 21 (P < 0.05). expression of ZO-1 and occludin in the jejunal mucosa The effects of early intervention with GOS on the pro- of the piglets. Significant dietary effects were observed tein expression of intestinal growth factors are shown in on the occludin mRNA expression levels (P < 0.05), and Fig. 3e-h. The results show that the protein expression a higher mRNA expression of occludin was observed in of GLP-2 and EGF were affected by diet. Piglets fed with piglets fed with GOS (P < 0.05) in contrast to those fed diets containing GOS showed significantly higher pro- with no GOS on d 8 (Fig. 5a-b). As shown in Fig. 5c-e, tein expression of GLP-2 and EGF than those fed with the protein expression of ZO-1 and occludin was af- no GOS (P < 0.05). fected by interactions between diet and age (P < 0.05). The protein expression of ZO-1 and occludin in the Disaccharidase activity and nutrient transporters jejunal mucosa also increased in the GOS group on d 8 As shown in Fig. 4a-c, lactase and sucrase activity were (P < 0.05). In addition, the protein expression of occludin affected by interactions between diet and age (P < 0.05), in the jejunal mucosa of the GOS piglets was higher and maltase activity was affected by diet. More specific- than that of the CON piglets. ally, piglets fed with diets containing GOS showed significantly higher maltase and sucrase activities (P < Intestinal immune factors 0.05) than those fed with no GOS on d 21, while piglets With respect to intestinal immune factors (Fig. 6), no fed with diets containing GOS showed significantly significant interactions between diet and age were higher lactase activity (P < 0.05) than those fed with no observed on the mRNA expression of IL-1β, IL-10, TLR4 GOS on d 8. and TNF-α (P > 0.05). However, significant interactions To assess gene expression related to the nutrient between diet and age were observed on the TGF-β transporters of jejunum, two kinds of genes were assayed mRNA expression levels (P < 0.05), and significant by q-PCR, as shown in Fig. 4d-e. It was demonstrated dietary effects were observed on the IL-12 mRNA that the mRNA expression of SGLT1 and GLUT2 was expression levels (P < 0.05). It was also determined that affected by interactions between diet and age (P < 0.05). GOS increased the mRNA expression of TGF-β (P < Piglets fed with diets containing GOS showed significantly 0.05) and reduced the mRNA expression of IL-12 on d 8 higher mRNA expression of GLUT2 (P < 0.05) than those (P < 0.05). fed with no GOS on d 8, while piglets fed with diets containing GOS showed significantly higher mRNA ex- Discussion pression of SGLT1 (P < 0.05) than those fed with no GOS In the present study, a neonatal piglet model was used on d 21. to study the effects of early intervention with GOS on growth performance and jejunal development during a D-lactic acid, diamine oxidase, tight junction genes, and week-long intervention period. By determining the protein expressions effects of early feeding strategies on the entire suckling The plasma D-lactic acid concentration and DAO activ- stage, the current experiment made it possible to evalu- ity in the suckling pigs are presented in Table 2. Signifi- ate the effects of GOS (from d 1 to d 7) on piglets’ cant interactions between diet and age (P < 0.05) were growth performance, jejunal morphology, disaccharidase Tian et al. Journal of Animal Science and Biotechnology (2018) 9:75 Page 6 of 11 activity, and barrier function at different ages. The re- sults suggested that GOS had significant effects on ADG, SI length, crypt depth, disaccharidase activity, tight junction expression, and gut permeability in suck- ling piglets. In our study, we referred to the dose of GOS used in rodent model to determine the appropriate dose of GOS for piglets. In previous studies, only the effect of in- creased abundance of Bifidobacterium has been reported in mice fed GOS of 0.26 g/(kg·d) [10]. While increased cecal total weight and wall weight have been reported in mice fed GOS of 1 g/(kg·d) [16]. Anthony et al. [15] found that rats fed GOS of 2.5 g/(kg·d) or 5 g/(kg·d) de- creased food consumption levels. Based on the reported results, the dose of 1 g GOS/kg weight was administered in our study. In addition, it has been reported that the natural oligosaccharide content of sows is approximately in the range of 0.05–0.1 g/dL [22]. According to the Ali- zadeh’s[23] research, we estimated that the total oligo- saccharide intake from the sow milk was about 0.3– 0.6 g/d when the piglets received sow milk of 600 mL/d during the 7 d after birth. In our study, piglets eventually intake GOS of 1.37–2.31 g/d (the initial BW of GOS group: 1.52 kg, the BW on d 7 of GOS group: 2.57 kg) with a consideration of the purity of GOS, about 4 times higher than the oligosaccharide intake solely from sow milk by the piglets. Previous studies have shown that the small intestine has demonstrated a significant increase in tissue mass and surface area of absorption in neonatal piglets [24–28]. For instance, the number of mucosal cells reportedly increased by 50% on the first day after birth and doubled on the third day after birth [28]. These studies indicated that the intestine of piglets developed fastest in the early stages of suckling piglets. Furthermore, it has been reported that GOS could increase cecal total weight and wall weight in mice [16]. Therefore, the pur- pose of this study was to improve intestinal development and increase the growth performance in the early stages of suckling piglets by supplementing with GOS. Consistent with our purpose, the SI length was significantly increased in GOS group on d 8. The increased SI length indicated that a significant increase in the area of the digestive and Fig. 2 The jejunal morphology of suckling piglets. Piglets assigned absorption of nutritious substances, thereby improving to CON (n = 6) and GOS (n = 6) received physiological saline and the growth performance of piglets. In addition, there have GOS solution for 7 d after birth, respectively. (a) Representative histological micrographs of jejunum in suckling piglets. The scale bar been several attempts to demonstrate the use of GOS as of jejunal morphology on d 8 was 500 μm, and the scale bar of potential promoters to enhance animal growth [29, 30]. jejunal morphology on d 21 was 200 μm. (b) Villus height, (c) Crypt Along the same lines as these studies, we have observed depth and (d) Villus height: crypt depth radio of jejunal morphology that early intervention with GOS could improve the BW in suckling piglets. Values are expressed as means ± SD. Bars and ADG of suckling piglets, which also consistent with assigned with different lower-case letters indicate a significant difference. CON: control group; GOS: GOS group our purpose. For suckling piglets, intestinal growth factors play a key role in the development of the intestine. For example, the GLP-1, GLP-2, EGF, and IGF-1 proteins Tian et al. Journal of Animal Science and Biotechnology (2018) 9:75 Page 7 of 11 Fig. 3 The expression of jejunal growth factors in suckling piglets. Piglets assigned to CON (n = 6) and GOS (n = 6) received physiological saline and GOS solution for 7 d after birth, respectively. a-d The relative mRNA expression of jejunal growth factors in suckling piglets. The values were -ΔΔCt calculated relative to the expression of β-actin with formula 2 . e-h The concentrations of jejunal growth factors in suckling piglets. Values are expressed as means ± SD. Bars assigned with different lower-case letters indicate significant differences. CON: control group; GOS: GOS group were able to increase the proliferation, differentiation, in the GOS group was higher than that in CON and apoptosis of intestinal epithelial cells [31, 32]. In this group, and the protein concentration of GLP-2 was study, we observed that the expressions of the intestinal consistent with the mRNA expression of the GCG. growth factors differed between GOS and CON The increased mRNA and protein expression of groups on d 8, but not on d 21. But interestingly, the GLP-2 could increase SI length through the stimula- expressions of intestinal growth factors in GOS pig- tion of epithelial cell antiapoptotic actions by activa- lets on d 8 were close to those in CON piglets and tors of the PI3K-Akt pathway [31]. The activation of GOS piglets on d 21. According to previous results, Akt in the intestinal mucosa has also been implicated the concentrations of growth factors in sow milk at in GLP-2-mediated epithelial glucose uptake [31]. In early lactation stage are higher than those at the late addition, IGF-1 has been identified as a major medi- lactation stage. This may cause the growth and devel- ator through which GLP-2 increases intestinal growth opment of the jejunum to reach the plateau stage at [34]. Also, high mRNA and protein expression of the late period of lactation [33]. And our dynamic GLP-2 may also be modulated by nutrient intake, es- change of daily weight gain confirms this speculation. pecially carbohydrate intake [35, 36]. Therefore, we Furthermore, we also observed that the mRNA ex- believe that GOS could improve the growth perform- pression of IGF-1 and GCG (the precursor of gluca- ance of suckling piglets via promoting jejunal devel- gon and other components is encoded by the GCG) opment and increasing carbohydrate intake. Fig. 4 The jejunal disaccharidase activity and mRNA expression of the glucose transport receptors in suckling piglets. Piglets assigned to CON (n = 6) and GOS (n = 6) received physiological saline and GOS solution for 7 d after birth, respectively. (a-c) The brush border enzyme activity of the jejuna in suckling piglets. (d-e) The relative mRNA expression of jejunal nutrient transporter in suckling piglets. The values were calculated -ΔΔCt relative to the expression of β-actin with formula 2 . Values are expressed as means ± SD. Bars assigned with different lower-case letters indicate significant differences. CON: control group; GOS: GOS group Tian et al. Journal of Animal Science and Biotechnology (2018) 9:75 Page 8 of 11 Table 2 Effects of galacto-oligosaccharides (GOS) on D-lactic acid and diamine oxidase (DAO) in suckling piglets . Items d 8 d 21 SEM P-value CON GOS CON GOS Diet Age Diet × Age a b b ab Plasma D-lactic acid, mg/L 13.13 12.42 12.62 12.81 0.15 0.116 0.661 0.010 a b b b Plasma DAO, units/mL 3.67 3.26 3.24 3.25 0.08 0.019 0.008 0.012 b a b b Jejunal mucosa DAO, units/mg mucosa 2.18 2.32 2.20 2.21 0.03 0.017 0.170 0.025 Piglets assigned to CON (n = 6), GOS (n = 6) received physiological saline and GOS solution for 7 d after birth, respectively Since the jejunum is the main organ for nutrient studies, the lactase activity on d 21 was lower than that absorption, we further analyzed jejunal morphology, on d 8, and the sucrase and maltase activities on d 21 disaccharidase activity, and carbohydrate transporters. In were higher than those on d 8. It is known that maltase the present study, early intervention with GOS signifi- and sucrase activities are important markers to evaluate cantly decreased the crypt depth on d 21, but it did not intestinal development [39, 40]. Hence, the increase of affect the villus height and villus height: crypt depth maltase and sucrase activities implied a certain rapid ratio in the jejunum. The most direct factor affecting maturation of the jejunum. In addition, the present study crypt depth was the change in the proliferation rate of showed that early intervention with GOS up-regulated intestinal stem cells [37]. In addition, decreased crypt lactase activity on d 8, and maltase, sucrase activities on d depth indicated that cell proliferation had decreased in 21. Furthermore, the up-regulated lactase activity on d 8, the GOS group. Villus height has been positively corre- and the maltase, sucrase activities on d 21 would promote lated with the number of cells present [37]. In this study, the polysaccharides in sow milk to be degraded into there was no difference in the villus height between the monosaccharides. This is conducive to the body absorbing two groups, which indicated that decreased cell prolifer- and utilizing the carbohydrates, thereby promoting intes- ation did not affect the growth of the jejunum. This may tinal maturity and host growth. These results therefore have been caused by the increased cell differentiation suggested that the piglets in the GOS group could utilize and the decreased cell apoptosis of the jejunum. Further- carbohydrates more efficiently than those in the CON more, disaccharidase activity is related to intestinal group. After hydrolyzation by disaccharidase, the carbohy- morphology. And the disaccharidase activity determines drates in a diet depend on a carbohydrate transporter to suckling piglets’ capacity for carbohydrate digestion and enter the bloodstream. In this study, the mRNA expres- transport. In previous studies, lactase activity was high sions of SGLT1 and GLUT2 were higher in the piglets with at birth but decreased with the age. However, sucrase GOS intervention than those in the piglets without GOS activity and maltase activity were low at birth, but their intervention, indicating an increased glucose transport activity gradually increased with the age until reaching rate of the intestine. Overall, these results indicate that stability [38]. Consistent with the findings of these early intervention with GOS enhances the degradation Fig. 5 The relative mRNA and protein expression of the jejunal tight junction in suckling piglets. Piglets assigned to CON (n = 6) and GOS (n =6) received physiological saline and GOS solution for 7 d after birth, respectively. (a-b) The relative mRNA expression of the jejunal tight junction in -ΔΔCt suckling piglets. The values were calculated relative to the expression of β-actin with formula 2 .(c) The blots of zonula occludens-1 (ZO-1), occludin, and β-actin of the jejunum mucosa in suckling piglets. (d-e) The relative protein expressions of the jejunal tight junction in suckling piglets. The value of protein expression was the ratio of the densitometry units of tight junction protein to β-actin. Values are expressed as means ± SD. Bars assigned with different lower-case letters indicate significant differences. CON: control group; GOS: GOS group Tian et al. Journal of Animal Science and Biotechnology (2018) 9:75 Page 9 of 11 Fig. 6 The jejunal immune function in suckling piglets. Piglets assigned to CON (n = 6) and GOS (n = 6) received physiological saline and GOS solution for 7 d after birth, respectively. (a-f) The relative mRNA expression of jejunal immune factors in suckling piglets. The values were -ΔΔCt calculated relative to the expression of β-actin with formula 2 . Values are expressed as means ± SD. Bars assigned with different lower-case letters indicate significant differences. CON: control group; GOS: GOS group rate of carbohydrates and the glucose transport rate in GOS intervention promoted the maturation of im- suckling piglets by modulating disaccharidase activity and mune function. In addition, several studies have the expression of glucose transport receptors. shown that the strength of the intestinal barrier was A good mechanical barrier can effectively prevent associated with enhanced piglet performance [43, 50]. bacteria, endotoxins, and other harmful substances In our study, the improvement of intestinal barrier from penetrating the intestinal mucosa, and in terms function was accompanied by increased growth per- of infrastructure, it functions as a tight junction be- formance. Therefore, increased barrier function may tween the intact intestinal epithelial cells and other ensure the absorption of nutrients and prevent bac- epithelial cells [41–43]. ZO-1 and occludin are main teria, endotoxins, and other harmful substances from transmembrane and nonmembrane proteins that entering the body through the intestinal mucosa [51]. form intercellular junctions between the epithelial cells [44, 45]. In addition, DAO and D-lactate serve Conclusion as indicators of intestinal integrity, as they are nor- In conclusion, the results obtained in the present mally presented in very small amounts in blood cir- study indicate that the increased in piglet growth culation. Increased plasma D-lactic acid levels and with GOS supplementation was associated with the serum DAO levels reflect changes in intestinal per- changes in expression of the genes and proteins in- meability, suggesting that the intestinal barrier func- volved in gut endocrine and barrier function, glucose tion has been damaged [46, 47]. Many studies have transporter and immune status. Further study is shown that GOS can reduce gut permeability and in- needed to investigate the exact mechanisms by which crease tight junction expression in vivo and in vitro GOS can promote intestinal development in suckling [48, 49]. Consistent with previous research results, piglets. our study shows that early intervention with GOS could improve the protein expression of ZO-1 and Additional file occludin in the jejunal mucosa on d 8. We also ob- served that plasma D-lactate and DAO decreased in Additional file 1: Table S1. Primer sequences for quantitative real-time theGOS group ond 8. Theseresults indicated that PCR analysis. (DOCX 17 kb) early intervention with GOS could enhance the bar- rier function of the jejunum. Furthermore, the im- Abbreviations provement of intestinal barrier function may imply ADG: Average daily gain; BCA: Bicinchoninic acid; DAO: Diamine oxidase; the improvement of intestinal immune function. DM: Dry matter; DP: Degree of polymerization; EGF: Epidermal growth factor; GAPHH: Glyceraldehyde phosphate dehydrogenase; GCG: Preproglucagon; Therefore, we analyzed the mRNA expression of in- GLP-2: Glucagon-like peptide-2; GLUT2: Glucose transporter type 2; flammatory factors. In this study, GOS increased the GOS: Galacto-oligosaccharides; HE: Hematoxylin and eosin; IGF-1: Insulin-like mRNA expression of TGF-β and reduced the mRNA growth factor 1; IGF-1R: Insulin-like growth factor 1 receptor; IL- 10: Interleukin-10; IL-12: Interleukin-12; IL-1β: Interleukin-1β; PBS: Phosphate expression of IL-12 on d 8. IL-12 is a buffer saline; PBST: Phosphate buffer, saline with Tween-2; pro-inflammatory cytokine, and TGF-β is an anti-in- PVDF: Polyvinylidene difluoride; SDS–PAGE: Sodium dodecyl sulfate– flammatory cytokine. According to the results, early polyacrylamide gel electrophoresis; SGLT1: Sodium glucose co-transporter 1; Tian et al. Journal of Animal Science and Biotechnology (2018) 9:75 Page 10 of 11 SI: Small intestine; TGF-β: Transforming growth factor-β; TLR4: Toll-like 11. Maathuis AJ, Heuvel EG, Schoterman MH, Venema K. Galacto- receptor 4; TNF-α: Tumor necrosis factor-α; ZO-1: Zonula occludens-1 oligosaccharides have prebiotic activity in a dynamic in vitro colon model using a (13) C-labeling technique. J Nutr. 2012;142:1205–12. 12. Sosa N, Gerbino E, Golowczyc MA, Schebor C, Gómez-Zavaglia A, Acknowledgements Tymczyszyn EE. Effect of Galacto-oligosaccharides: Maltodextrin matrices on The authors thank the National Center for International Research on Animal the recovery of lactobacillus plantarum after spray-drying. Front Microbiol. Gut Nutrition for financial support. 2016;7:584. 13. Varasteh S, Braber S, Garssen J, Fink-Gremmels J. Galacto-oligosaccharides Funding exert a protective effect against heat stress in a Caco-2 cell model. 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Published: Oct 18, 2018

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